Name: the corneal micropocket assay: a model of angiogenesis in the mouse eye
Uploaded: 2014-08-16T17:00:00
Description: Watch this Scientific Journal Video about The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye at JoVE.com

我们感谢克里斯汀·约翰逊的图形工作。

所有涉及动物的协议是在波士顿儿童医院提交并经实验动物管理和使用委员会，并按照协会评估实验动物照护及认可委员会（AAALAC）的建议进行的。请务必使用消毒仪器和无菌techniqure在执行的过程。 1，制备的颗粒<ol><li>称取硫糖铝10毫克，并用无菌展平铲60毫克海昌的。发生在不同的离心管。 </li><li>将1厘米见方的块网格进入无菌10cm皿。反转所以目休息的浅盖。抛开。 </li><...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laminin+and+basement+membrane+in+the+morphological+differentiation+of+human+endothelial+cells+into+capillary-like+structures.">Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.</a> J. Cell Biol. 107, 1589-1598 (1988).</li><li>Montesano, R., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-promoting+phorbol+esters+induce+angiogenesis+in+vitro.">Tumor-promoting phorbol esters induce angiogenesis in vitro.</a> Cell. 42, 469-477 (1985).</li><li>Korff, T., Augustin, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+endothelial+cells+in+multicellular+spheroids+prevents+apoptosis+and+induces+differentiation.">Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation.</a> J. Cell Biol. 143, 1341-1352 (1998).</li><li>Nicosia, R. F., Tchao, R., Leighton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histotypic+angiogenesis+in+vitro:+light+microscopic,+ultrastructural,+and+radioautographic+studies.">Histotypic angiogenesis in vitro: light microscopic, ultrastructural, and radioautographic studies.</a> In Vitro. 18, 538-549 (1982).</li><li>Wong, K. H., Chan, J. M., Kamm, R. 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K., Schlenger, K., Hockel, M., Yuan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+angiogenesis+assays:+progress+and+problems.">Quantitative angiogenesis assays: progress and problems.</a> Nat. Med. 3, 1203-1208 (1997).</li><li>Rogers, M. S., Birsner, A. E., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cornea+micropocket+angiogenesis+assay.">The mouse cornea micropocket angiogenesis assay.</a> Nat. Protoc. 2 (10), 2545-2550 (2007).</li><li>Gimbrone, M. A., Leapman, S. B., Cotran, R. S., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+dormancy+in+vivo+by+prevention+of+neovascularization.">Tumor dormancy in vivo by prevention of neovascularization.</a> J. Exp. Med. 136, 261-276 (1972).</li><li>Gimbrone, M. A., Cotran, R. S., Leapman, S. B., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+growth+and+neovascularization:+an+experimental+model+using+the+rabbit+cornea.">Tumor growth and neovascularization: an experimental model using the rabbit cornea.</a> J. Natl. Cancer Inst. 52, 413-427 (1974).</li><li>Rohan, R. M., Fernandez, A., Udagawa, T., Yuan, J., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+heterogeneity+of+angiogenesis+in+mice.">Genetic heterogeneity of angiogenesis in mice.</a> FASEB J. 14 (7), 871-876 (2000).</li><li>Chang, L. 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有在执行一个成功的角膜检测的几个关键步骤。首先是制作能够被植入并刺激血管均匀的颗粒。粒料制备中最重要的部件是：1）使用无载体的生长因子; 2）确保生长因子与硫糖铝和海昌和3个好混合物）迅速但仔细一动最终混合物从Eppendorf管到的颗粒投网。我们建议进行“虚拟”颗粒无生长因子练技术。球团应该会出现白色和粉化，并大致呈正方形。这包括明确的区域的任何颗粒，应排除在外，?...

血管生成的过程中，新血管的形成，形成预先存在的那些，是非常复杂的，并通过控制不同的步骤血管出芽和形态发生的几内源性因子的调节。血管生成是由于亲和抗血管生成因子之间的转变，平衡，通常保持在静止状态下的脉管系统的平衡被触发。血管发生在成人中发生一定的生理条件，如女性卵巢周期期间或在维修过程中如伤口愈合和组织再生。然而，它也是几种病状，包括恶性肿瘤，自身免疫性疾病和炎症性疾病的标志。血管生成的这些生理和病理条件下参与使得它对于研究和治疗的一个有吸引力的目标的一个重要课题。 由于血管发生的复杂性和几种策的参与LLS和因素的过程中，包括内皮细胞，周细胞，循环细胞和基质细胞， 在体外模型中仍是有限的，不能概括的唯一的体内微环境。 在血管生成的体外测定法的主要在很大程度上集中于观察对内皮细胞的直接影响和控制的条件下测量在血管生成过程中的某些步骤。这些测定法包括内皮细胞的增殖1，迁移2，网络形成三管形成4的量化，并从球状体5发芽。 体外模型，不同的是在体外的人，都比较复杂并包含多种组织类型的细胞，一个例子是主动脉环测定法6。然而，像其他系统，它不能在体内存在的捕获循环细胞和贡献内皮细胞的天然基质。尝试研究根据流量的血管生成模拟体内环境使用微流体系统7中执行，但即使是这些试验中，虽然大大改善，仍无法考虑到所有存在于体内的车厢。 由于在体外和离体血管生成模型中，限制在体内模型仍然是血管生成的研究更可靠的选择。这些模型的实例包括透明腔室，“窗口”，即允许的生长血管显微镜8下的可视化的植入剂，注射皮下植入物，如基质胶和血管形成中的正常组织，如小鼠耳和鸡胚绒毛尿囊膜（CAM ）。然而，最可接受的和定量的体内血管生成的模型之一是在此描述的角膜微囊新血管形成测定法，其利用了自然股骨头缺血性角膜作为一个“屏”的可视化和评估新的血管生长9。 在这里，我们描述了角膜微囊测定作为开发中的小鼠。最初的模型被用来测量在兔角膜的非特异性血管生成的刺激。这是通过引入肿瘤块放入兔眼的前房的前房进行测定和肿瘤诱导的血管生成11。 然而，该法后来演变来研究特定生长因子的影响10，以更好地说明和规范血管生成作用。为了释放生长因子在眼内，来代替组织中含有已知量的血管生成生长因子的缓释颗粒。纯化的重组血管生成蛋白如的bFGF或VEGF的可用性使他们的血管生成modulaters 12的具体目标使用。最初，该测定是主要用于在兔，这是更容易使用，因为它们的大小，但后来的模型被翻译成小鼠正常工作;更小和更便宜的动物模型。从兔转移到小鼠的效果是：能够使用遗传操作的动物，从而产生一个新的研究领域进入遗传组分影响血管13的一个重要优点。除了角膜法的研究血管生成的多种可接受的使用，其他的生物学过程，也被用改性试验研究。例如，淋巴管生成的研究，通过低剂量的bFGF球团这使淋巴管，通过特定的分子标记14的可视化的植入能够成为可能。此外，该测定中提供了评估血管生成15辐射的影响的装置。 在总结中，角膜微囊血管生成分析是定量的，代表roducible，血管生成的体内灵活的评估。该测定的主要优点是，背景血管测量是不必要的，因为血管生长在天然无血管组织。我们在这里描述这个实验的详细协议，并讨论可能出现不同的情况。该试验由3离散区段。在这里，我们将描述的生长因子包括粒料用于量化所得到的新血管生长的制备，随后的外科手术植入，最后的方法。

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			<td height="21" style="height:21px;width:216px;">Section 1: Pellet preparation</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucralfate</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">#S0652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydron (aka Poly(2-Hema))</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">#P3932-10G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol</td>
			<td style="width:111px;">Pharmco Products Inc</td>
			<td style="width:119px;">#111000200CSGL</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Growth factors:</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Must be carrier-free (no bovine serum albumin(BSA))</td>
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			<td height="21" style="height:21px;width:216px;">&#160;Fibroblast growth factor (FGF)</td>
			<td style="width:111px;">PeproTech</td>
			<td style="width:119px;">#AF-100-18B</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;width:216px;">Vascular endothelial growth factor (VEGF)</td>
			<td style="width:111px;">R &#38; D Systems</td>
			<td style="width:119px;">#293-VE-050/CF</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">35 mm dish</td>
			<td style="width:111px;">Becton-Dickson</td>
			<td style="width:119px;">#353001</td>
			<td>Used for storage of pellets</td>
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			<td height="21" style="height:21px;width:216px;">10 cm petri dish</td>
			<td style="width:111px;">VWR</td>
			<td style="width:119px;">#25384-342</td>
			<td>Used as work surface for preparing pellets</td>
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			<td height="21" style="height:21px;width:216px;">Mesh</td>
			<td style="width:111px;">Sefar America</td>
			<td style="width:119px;">#03--300/51</td>
			<td>300 um nitex nylon, cut into cm square pieces and sterilzed in autoclave</td>
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			<td height="21" style="height:21px;width:216px;">Spatulas</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:119px;">#21-401-10</td>
			<td>Use tapered end of one to break up pellet mixture. Bend tapered end of other to help remove mixture from microcentrifuge tube.</td>
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			<td height="21" style="height:21px;width:216px;">Microcentrifuge tubes</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:119px;">#05-408-146</td>
			<td>One for hydron, one for sucralfate</td>
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			<td height="21" style="height:21px;width:216px;">Jewelers forceps, #5</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#2315E</td>
			<td>Need 2 for pulling mesh apart</td>
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			<td height="42" style="height:42px;width:216px;">Centrifugal evaporator</td>
			<td style="width:111px;">ThermoSavant</td>
			<td style="width:119px;">DNA110 SpeedVac</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Section 2: Surgical implantation of pellets</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Operating microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">2.5% Avertin</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>General anesthetic</td>
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			<td height="42" style="height:42px;width:216px;">Proparacaine hydrochloride ophthalmic solution 0.5%</td>
			<td style="width:111px;">Falcon</td>
			<td>NDC# 6131401601</td>
			<td>Eye anesthetic</td>
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			<td height="42" style="height:42px;width:216px;">Triple Antibiotic Ophthalmic Ointment</td>
			<td style="width:111px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">NDC# 2420878055</td>
			<td>Contains neomycin, polymixin and bacitracin</td>
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			<td height="21" style="height:21px;width:216px;">Ophthalmic microknife, 5 mm</td>
			<td style="width:111px;">Surgistar</td>
			<td style="width:119px;">#924501</td>
			<td>30 degree angle</td>
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			<td height="21" style="height:21px;width:216px;">von Graef knife</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#3401E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jewelers forceps, #1</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#2301E</td>
			<td>Must be blunted with sharpening stone for proptosing eye</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jewelers forceps, #5</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#2305E</td>
			<td>For picking up pellets and placing on eye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small curved scissors</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#5636E</td>
			<td>For trimming whiskers</td>
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			<td height="21" style="height:21px;width:216px;">Gauze</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>For blotting eye after proparacaine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Section 3: Grading of Corneal Neovascularization</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2.5% Avertin</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>General anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Slit lamp</td>
			<td style="width:111px;">Nikon</td>
			<td style="width:119px;">FS-2</td>
			<td>Needs an ocular with a reticule to assist in measuring</td>
		</tr>
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</table>

the corneal micropocket assay: a model of angiogenesis in the mouse eye

小鼠角膜微囊法是一个强大的和定量的体内试验，用于评估血管生成。通过使用含有该触发血管生长的整个自然股骨头缺血性角膜特异性生长因子标准化的缓释小球，血管生成可被测量和量化。在该测定中的血管生成反应是在数天的过程中产生的，这取决于所用的生长因子的类型和剂量。新血管形成的诱导通常由任一碱性成纤维细胞生长因子（bFGF）或血管内皮生长因子（VEGF）引起的。通过组合这些生长因子与硫糖铝和昌（聚-HEMA（聚（2 - 羟乙基甲基丙烯酸酯）））和铸造的混合物制成丸粒，它们可以通过外科手术植入在小鼠眼。这些颗粒均匀缓慢释放的生长因子在五，六天（bFGF的或VEGF分别为），使所需的船只区域Q足够的血管生成反应uantification用裂隙灯。此测定法可用于不同的应用，包括血管生成调节剂的药物或治疗方法，以及影响血管生成不同的遗传背景之间的比较进行评价。练此法后，一个熟练的研究者可以在每眼不到5分钟植入颗粒。

典型的结果为bFGF和VEGF的小球在正常低血管C57BL / 6J小鼠示于图2A和B中 。 图2E示出了血管区域的正态分布（VA）在C57BL / 6J应变与不同剂量的这些生长因素。 80纳克的bFGF粒料通常导致的约为2.0 mm 2的VA，虽然在一系列1.8-2.4毫米2的值是可以接受的。 200 ng的血管内皮生长因子颗粒通常导致0.7mm左右2的响应，范围可以接受的0.6-0.9毫米2...

Watch this Scientific Journal Video about The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye at JoVE.com

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

角膜微囊含量：血管生成在小鼠眼模型

该协议描述为开发在小鼠角膜微囊测定。

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Research

JoVE Journal

Neuroscience

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

在阿尔茨海默氏症小鼠模型神经炎斑检测

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

在小鼠大脑的各个区域端粒酶活性：非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...